Our research has three main long-term objectives: (i) Detailed analyses of the structure/function of nucleic acid analogs, for the design of the next generation of antisense oligonucleotide (AON) and RNAi therapeutics with potential anticancer, antiviral and anti-inflammatory indications; (ii) Determination of crystal structures of nucleic acid analogs studied in the context of etiology (origin of nucleic acid structure); (iii) Structural investigation of DNA fine structure, for assessing the relative influence of sequence and cations on conformation. The main tool we will use for determining the three dimensional structures is X-ray crystallography. In the first project, two key features of antisense compounds will be given particular attention: RNA affinity and the origins of antisense-RNA substrate binding and recognition by E.coli RNase H. The crystal structures of selected 2nd and 3rd generation AON and 1st generation siRNA modifications will be determined. The structural data will be correlated with their thermodynamic stabilities, nuclease resistances, susceptility to E. coli RNase H degradation (AONs) and overall efficacy, in collaboration with Isis Pharmaceuticals Inc. (AONs) and Alnylam Pharmaceuticals (siRNAs). In addition, we will seek to answer the question why RNase H binds double-stranded RNA (dsRNA), but does not cleave RNA strands paired to RNA (unlike those paired to DNA), by solving the crystal structures of DNA:RNA-RNase H and dsRNA-RNase H complexes. In the second project, we will analyze the crystal structures of alternatives to the natural nucleic acids with shorter backbones; i.e., threofuranosyl and lyxopyranosyl nucleic acid (TNA and LPNA, resp.), using either partially or all-modified oligonucleotides. The third project is an extension of our previous efforts to maximize the resolution of DNA crystal structures, to gain insight into the relative influence of sequence and metal cations on DNA duplex conformation. We will use neutron scattering, to analyze the structure of a representive oligodeoxynucleotide, and to visualize fully and partially occupied alkali metal ion binding sites. The specific aims of this research are: (1) Crystallographic analyses of 2nd and 3rd generation AON modifications, and correlation of the structures with existing in vitro and in vivo functional data; (2) Crystallographic analyses of siRNA modifications that are tolerated by the RNAi pathway, to improve RNA affinity and nuclease resistance of siRNA; (3) Structural studies of AONs (arabino nucleic acids) that, in complex with RNA, are substrates of RNase H; (4) Crystal structures of DNA:RNARNase H and dsRNA-RNase H complexes; (5) Crystallographic analyses of artificial nucleic acid pairing systems; and (6) Neutron diffraction studies of a DNA duplex and high-resolution X-ray structures of DNAs carrying methylphosphonates.